CN111467472B - Immunoregulation microsphere preparation targeting tumor-associated macrophages and preparation method and application thereof - Google Patents
Immunoregulation microsphere preparation targeting tumor-associated macrophages and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an immunoregulation microsphere preparation targeting tumor-associated macrophages, and a preparation method and application thereof. The microsphere preparation is a double-targeting preparation combining macrophage targeting peptide and B cell lymphoma 6 factor inhibitory peptide; loading B cell lymphoma 6 factor inhibitory peptide into liposome to obtain B cell lymphoma 6 factor inhibitory peptide-liposome solution; and combining the B cell lymphoma 6 factor inhibitory peptide-liposome solution with macrophage targeting peptide to obtain the final product. The invention combines the targeting peptide of the macrophage with the intracellular signal molecule inhibitor for the first time, and the targeting peptide is loaded on the nano-microsphere to prepare the immune preparation which is directionally transferred to the tumor microenvironment. In addition, the invention discovers for the first time that the preparation can obviously reduce the proportion and the number of macrophages in the lung cancer tumor, reverses the stem cell-like phenotype, can obviously inhibit the size and the weight of the lung cancer tumor, and has obvious anti-tumor effect.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an immunoregulation microsphere preparation targeting tumor-associated macrophages, a preparation method thereof and application thereof in preparing anti-tumor products.
Background
Tumors are currently globally common healthThe problem is a great problem threatening the health of human beings, the morbidity and the mortality of the problem are increased year by year, and the slowing or inhibiting of the development of the tumor becomes a main target of the current tumor treatment. Existing studies have established that the development of tumors is closely related to the Tumor Microenvironment (TME) in which they are located. TME not only provides necessary nutrients, growth conditions and physical matrix for tumor growth, but also transforms immune cells such as lymphocytes, macrophages and the like infiltrated therein through transformation and domestication so as to cause phenotype, function transformation or effector cell depletion, thereby forming a local small environment (niche) which is beneficial to tumor immune escape and promotes the growth of the tumor. Based on this, currently, tumor immunotherapy such as PD-1 immune checkpoint inhibitor and CAR-T, which aims at relieving immune suppression, is receiving wide attention, and is considered as a new tumor treatment technology with important application prospects and great potential, in addition to traditional tumor treatment methods such as surgery, chemotherapy, and radiotherapy. Nevertheless, current tumor immunotherapy approaches mostly focus on T cells, whose activation and function is performed by antigen presenting cells such as Dendritic Cells (DCs) and macrophages
And the like, so that the immunoregulation medicament and the treatment method taking the macrophage as the target are becoming a new research direction of tumor immunotherapy.
Tumor-associated macrophages (TAMs) are a major component of the tumor microenvironment, with a proportion of tumor tissue that can even reach more than 50%. A great deal of clinical evidence indicates that the number of TAMs has a close relationship with tumor occurrence, development, metastasis, invasion and the like, is negatively related to the tumor treatment effect and the prognosis of patients, and has an inverse relationship with the curative effect of the current tumor immunotherapy methods such as PD-1 inhibitors. The research results of our and other researchers show that the TAMs are differentiated into cell subtypes with specific phenotypes and functions under the action of tumors, show that the cell proliferation, survival and self-replication capacities are remarkably enhanced, the levels of secretory immunosuppression and cancer-promoting cytokines are remarkably increased, have the action of directly or indirectly promoting the formation and development of the tumors, and are very attractive tumor immunotherapy targets. Currently, some advances have been made in tumor immunotherapy strategies against TAMs, including removal of TAMs, interference with TAMs function, reprogramming of TAMs phenotype, but there are still many problems to be solved. For example, many treatment methods based on the objective of eliminating TAMs lack tumor targeting and have poor curative effect due to low permeability to the tumor microenvironment. More importantly, since macrophages are the core component of the innate immunity of the organism and have important functions of resisting infection, immunoregulation, tissue repair, maintaining organism balance and the like, nonspecific clearance or universal reprogramming of macrophage phenotype function is undoubtedly unfavorable for ensuring the safety and effectiveness of anti-tumor, and TAMs with clear mechanism, specific action targets and immunosuppression effect are sought.
B-cell lymphoma 6 factor (B cell lymphoma 6, Bcl6) is a transcription factor with multiple regulatory activities. The structure of the zinc-containing molecular marker comprises an N-terminal BTB/POZ region and 6 kruppel-like zinc finger structures at the C terminal. Bcl6 regulates transcription and expression by binding to a specific DNA sequence in the promoter region of a target gene. The Bcl6 target gene relates to a plurality of key molecules of cell activation, differentiation, development, apoptosis, proliferation, cell energy metabolism and the like, and participates in a plurality of physiological and pathological processes of immunity, inflammation, tumor, development and the like. Earlier in this study, Bcl6 was inducibly highly expressed in TAMs and caused them to retrodifferentiate into a macrophage phenotype with stem cell-like, tumor-memory promoting properties. Research shows that Bcl6 has the functions of promoting macrophage proliferation, resisting apoptosis, self-replication and secreting immune inhibiting cell factor and is important reason for the long-term survival, increase of TAMs and limitation of tumor eliminating capacity in tumor microenvironment. Interference with Bcl6 expression has shown significant in vivo anti-tumor effects, and therefore, Bcl6 is expected to be a potential molecular target for anti-tumor immunotherapy targeting tumor-associated macrophages to reverse immunosuppression.
Disclosure of Invention
The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide an immunoregulation microsphere preparation targeting tumor-related macrophages, aiming at the defects of effective targets, technical ideas and method means aiming at targeting tumor-related macrophages and specifically reversing immunosuppression in the prior anti-tumor immunotherapy, wherein the immunoregulation microsphere preparation has the advantages of cell and molecule double targeting, directional transfer to a tumor microenvironment and effective improvement of anti-tumor immune effect.
The technical problem to be solved by the invention is to provide a preparation method of the immunoregulation microsphere preparation for targeting tumor-associated macrophages.
The technical problem to be solved by the invention is to provide the application of the immunoregulation microsphere preparation targeting tumor-associated macrophages in preparing anti-tumor products.
In order to solve the technical problems, the invention discloses an immunoregulation microsphere preparation targeting tumor-associated macrophages, which is a double-targeting immunoregulation microsphere preparation taking nano microspheres as a carrier and combining macrophage targeting peptide (Mp) and B cell lymphoma 6 factor (Bcl6) inhibitory peptide.
Wherein the amino acid sequence of the macrophage targeting peptide (Mp) is YEQDPWGVKWWY; the amino acid sequence of the B cell lymphoma 6 factor (Bcl6) inhibitory peptide is GEGIEHISR.
The preparation method of the immunoregulation microsphere preparation targeting the tumor-associated macrophages is also within the protection scope of the invention.
Wherein, the preparation method comprises the steps of loading Bcl6 inhibitory peptide (Bi) on the surface of liposome to obtain a Bi-liposome solution; combining the Bi-liposome solution with the Mp, and mixing the two solutions under the acidic condition of pH2-4 to obtain the final product.
The preparation method of the Bi-liposome solution comprises the steps of dissolving B cell lymphoma 6 factor inhibitory peptide in PBS buffer solution, adding phospholipid film, vibrating and hydrating at room temperature, homogenizing the particle size, dialyzing, and removing the free state.
The preparation method of the phospholipid film comprises the steps of dissolving phospholipid and cholesterol in chloroform, and removing a dry solvent to obtain the phospholipid film; wherein the dosage ratio of the phospholipid, the cholesterol and the trichloromethane is 60 mg: 10 mg: 4 mL.
Wherein the dosage ratio of the B cell lymphoma 6 factor inhibitory peptide to the PBS buffer solution is 1-4mg/mL, preferably 2.5 mg/mL; the amount of the phospholipid membrane is controlled so that the molar ratio of the phospholipid to the B-cell lymphoma 6 factor inhibitory peptide is 40:1 to 8:5, preferably 8: 1.
Wherein the time of shaking hydration is 30 min.
Wherein, the homogenized particle size is ultrasonic by a probe, and is preferably 200W multiplied by 10 times.
Wherein, the combination of the B cell lymphoma 6 factor inhibiting peptide-liposome solution and the macrophage targeting peptide comprises the following steps:
(1) dissolving macrophage targeting peptide in PBS buffer solution, and adjusting pH of the solution to 2-4, preferably 3.5, by using HCl;
(2) and (2) mixing the solution obtained in the step (1) with a B cell lymphoma 6 factor inhibitory peptide-liposome solution, adjusting by HCl to keep the pH value of the mixture at 3.5 acidic condition, and shaking for 30min at room temperature to obtain the compound.
In step (1), the dosage ratio of the macrophage targeting peptide to the PBS buffer solution is 1-4mg/mL, preferably 3.3 mg/mL.
In the step (2), the dosage of the solution obtained in the step (1) and the B cell lymphoma 6 factor inhibiting peptide-liposome solution is controlled to ensure that the molar ratio of the macrophage targeting peptide to the phospholipid is 1:50-1:200, preferably 1: 50.
The application of the immunoregulation microsphere preparation targeting tumor-related macrophages in preparing anti-tumor products is also within the protection scope of the invention; preferably, the tumor is lung cancer.
The anti-tumor microsphere preparation specifically eliminates tumor-related macrophages through the immunoregulation microsphere preparation, inhibits the dry transformation and proliferation capacity of the tumor-related macrophages, improves the effect of immunosuppression on a tumor microenvironment, can obviously inhibit the growth and development of in vivo tumors, and has potential application value in prevention and treatment of tumors such as lung cancer.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the invention combines the targeting peptide of the macrophage with the intracellular signal molecule inhibitor for the first time, and the targeting peptide is loaded on the nano-microsphere to prepare the immune preparation which is directionally transferred to the tumor microenvironment.
(2) The experimental result shows that the preparation can obviously inhibit the size and the weight of the lung cancer tumor and has obvious anti-tumor effect.
(3) The invention discovers for the first time that the preparation can obviously reduce the proportion and the number of macrophages in lung cancer tumors, reverses the stem cell-like phenotype of the lung cancer tumors, and provides a strategy for immunotherapy based on tumor macrophage-associated cells.
(4) The small molecule preparation provided by the invention can specifically target tumor macrophages on one hand, and promote the tumor macrophages to phagocytose the tumor macrophages into cells on the other hand due to the characteristic that the macrophages have exogenous particles. Compared with single target molecule inhibitory peptide or small interfering RNA, the microsphere particle has more definite cell positioning and better penetrability, and has the advantages that other non-targeted immune preparations and treatment methods do not have in the aspects of improving local curative effect and reducing systemic influence.
Drawings
FIG. 1 is an electron micrograph of a liposome loaded with Bcl6 inhibiting peptide.
FIG. 2 shows the preparation and characterization of TAM-Bi.
FIG. 3 is a representation of entry of microspheroidal particles into macrophages.
FIG. 4 shows the expression of Bcl6 in TAMs and its effect on the dryness of TAMs. Figure 5 is the effect of TAM-Bi on mouse tumor size and weight, where P < 0.001.
Figure 6 is the effect of TAM-Bi on the number and proportion of tumor associated macrophages, where P < 0.01.
Figure 7 is a graph of the effect of TAM-Bi on tumor associated macrophage spheronization, where P < 0.01.
FIG. 8 is a graph showing the effect of TAM-Bi on the expression of sternutators by tumor-associated macrophages.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
Example 1
1. Experimental Material
1.1 experimental drugs and reagents: soya lecithin and cholesterol (biochemical family of Shanghai Meclina); bcl6 inhibitory peptide GEGIEHISR and macrophage targeting peptide YEQDPWGVKWWY (gill biochemical, inc.); reverse transcription kit (Thermo Fisher Scientific); SYBR Green dye (Thermo Fisher Scientific); flow through antibodies (eBioscience); bcl6 antibody (Cell Signaling Technology); bcl6 Small interfering RNA (Shanghai Jima biopharmaceutical technologies, Inc.).
1.2 experimental animals and breeding: C57/BL6 mice, 6-9 weeks old, supplied by Nanjing university model animals; complete nutrition pellet feed: provided by cooperative medical biotechnology limited of Jiangsu province; feeding conditions are as follows: room temperature 20 plus or minus 2 deg.c, humidity 55-65%, alternate light and shade, proper light intensity, and good ventilation and cleanness.
2. Experimental methods
2.1 construction and characterization of macrophage targeting microsphere preparation.
60mg of phospholipid, 10mg of cholesterol and 10mg of Bcl6 inhibitory peptide are weighed according to the mol ratio of 1:1.3:8 of Bcl6 inhibitory peptide, cholesterol and phospholipid. Dissolving phospholipid and cholesterol in 4mL of chloroform, and performing rotary evaporation at room temperature to remove dry solvent to obtain the phospholipid film. Bcl6 inhibitory peptide was dissolved in 4mL PBS buffer, added to phospholipid membrane, shaken at room temperature for 30min to complete hydration, sonicated with probe 200 Wx 10 times, and homogenized for particle size. The size and the potential of the particles are detected by a Malvern particle size analyzer, the size and the form are detected by a transmission electron microscope, and the encapsulation efficiency is detected. Taking a proper amount of Bcl6 inhibitory peptide-liposome in a 3000D dialysis bag, and dialyzing for 5h by using PBS as a dialysis medium to remove free peptide to obtain a Bcl6 inhibitory peptide-liposome solution.
Weighing 3.3mg of macrophage targeting peptide according to the molar ratio of the macrophage targeting peptide to the phospholipid of 1:50, dissolving in 1mL of PBS buffer solution, and adjusting the pH of the solution to 3.5 by HCl, thus obtaining the macrophage targeting control preparation (TAM-Con).
Mixing the solution with the prepared Bcl6 inhibitory peptide-liposome solution, adjusting with HCl to maintain pH at 3.5 acidic condition, and shaking at room temperature for 30min to obtain macrophage targeting immunoregulation preparation (TAM-Bi).
0.5mL of TAM-Con or TAM-Bi prepared as described above was dissolved in 2mL of PBS buffer solution, and 2 portions of each 0.5mL of TAM-Con or TAM-Bi were added to a 4mL centrifuge tube, one portion was diluted with 2mL of pure water to prepare a control, and the other portion was added with 2mL of 0.2mol/L sodium iodide solution and mixed and shaken for 5 min. The emission spectrum intensity of tryptophan in different groups is respectively measured by taking 280nm as an excitation wavelength, and the binding condition of the targeting peptide and the Bcl6 inhibiting peptide is tested by an iodide ion fluorescence quenching method.
2.2 construction of mouse lung cancer model.
20C 57/BL6 mice (6-8 weeks old) were selected and randomized into control and treatment groups of 2 groups of 10 mice each. Will be 1 × 106Lewis lung carcinoma cells were mixed well with 500. mu.g of macrophage targeting microsphere preparation not loaded with Bcl6 inhibitory peptide (TAM-Con, control group) and loaded with Bcl6 inhibitory peptide (TAM-Bi, treatment group), respectively, and injected subcutaneously into the right hind dorsal of each mouse. And injecting the liposome microspheres into tail vein every other day 3-19 days after molding. Mice were sacrificed 4 weeks later for subsequent functional analysis.
2.3 Effect of macrophage targeting immune preparation on lung cancer tumor.
The established tumor-bearing mice were sacrificed by 5 per group, the tumors on the right lumbar dorsal side were dissected off, and the weight of each tumor was weighed, and the tumor volume was determined by measuring the length (l) and width (w) and calculating the volume (V ═ l/2 × w 2).
2.4 characterization of entry of microspheroidal particles into macrophages.
Weighing 6.5mg of rhodamine, 60mg of phospholipid and 10mg of cholesterol according to the molar ratio of 1:6 of rhodamine to phospholipid. Phospholipid and cholesterol were dissolved in 4mL of chloroform, the solvent was removed by rotary evaporation at room temperature, and rhodamine and 10mg of Bcl6 inhibitory peptide were dissolved in 4mL of PBS buffer. Adding the PBS buffer solution into phospholipid film, shaking for 30min to make hydration complete, performing ultrasonic treatment with probe for 200W × 10 times, and homogenizing particle size. And (3) putting a proper amount of rhodamine-liposome into a 3000D dialysis bag, and dialyzing for 5 hours by using PBS as a dialysis medium to remove free peptide, thus obtaining the rhodamine-Bcl 6 inhibitory peptide-liposome solution.
Mixing the solution with the prepared rhodamine-Bcl 6 inhibitory peptide-liposome solution, adjusting by HCl to keep the pH value at 3.5 acidic condition, and shaking at room temperature for 30min to obtain the rhodamine-loaded macrophage targeted immunoregulation preparation.
The established tumor-bearing mice are injected with the rhodamine-loaded macrophage targeted immunoregulation preparation by tail vein every day for 3 consecutive days from 14 days after the model building, 3 mice are taken out after 1 day and killed, tumors on the right waist and back are stripped, the mice are cut into pieces, 2 times of volume of collagenase A (1mg/mL) is added into the cut tumor tissues of each mouse, the enzyme amount is 0.223U/mg, the enzyme activity is defined to act on the natural collagen under the conditions of 37 ℃ and ph7.5, and the polypeptide released from the collagen per hour is equivalent to the ninhydrin coloration with 1 micromole leucine amount as an activity unit (U). Mixing the above solutions, digesting in a shaker at 37 deg.C and rotation speed of 180 rpm for 1 hr to convert more than 95% of tissue into cell suspension, filtering the cell suspension to remove impurities, mixing, and counting. Take 1X 106Adding flow antibody anti-mouse F4/80 and CD11b into each cell, incubating for 1 hour in a dark place, and detecting F4/80 in single cell suspension by flow cytometry+CD11b+The ratio of rhodamine positive cells in the cells, namely macrophages.
2.5 Bcl6 expression of TAMs and its effect on dryness of TAMs.
The lung cancer model is constructed by taking 4C 57/BL6 mice (6-8 weeks old) and killing all the mice after 4 weeks, stripping off tumors on the right waist and back, shearing, adding collagenase A (1mg/mL) with the volume 2 times of the tumor tissue of each mouse, wherein the enzyme amount is 0.223U/mg, the enzyme activity is defined as acting on the natural collagen at 37 ℃ and ph7.5, and the amount of polypeptide released from the collagen per hour is equivalent to ninhydrin coloration and 1 micromole leucine (U) as an active unit. And (3) uniformly mixing the solution, placing the mixture in a shaking table at the temperature of 37 ℃ and the rotating speed of 180 revolutions per minute for digestion for 1 hour, converting more than 95 percent of tissues into cell suspension, filtering the cell suspension to remove impurities, and uniformly mixing to obtain the tumor tissue cell suspension. Each one is preparedThe cells obtained from the mice are inoculated in a culture dish of 10cm, placed at 37 ℃ and kept stand for 40min, and the culture supernatant is removed, and the remained adherent cells are TAMs. The expression of Bcl6 in TAMs was detected by using western blot. Take 1X 106And (3) transfecting small interfering RNA (experimental group) and empty vector (control group) of Bcl6 into cells of RAW264.7 macrophages, stimulating the cells for 24 hours by using lung cancer cell culture supernatant, collecting the cells, extracting RNA, and detecting the expression condition of the dryness related factor by adopting a qPCR method.
2.6 Effect of macrophage targeting immune formulations on macrophage number and proportion.
The established tumor-bearing mice are taken and killed by 5 mice each, the tumor on the right waist and back is stripped, the mice are cut into pieces, 2 times of volume of collagenase A (1mg/mL) is added into the cut tumor tissue of each mouse, the enzyme quantity is 0.223U/mg, the enzyme activity is defined as acting on natural collagen at 37 ℃ under the ph7.5 condition, and the polypeptide released from the collagen per hour is equivalent to the ninhydrin coloration with 1 micromole of leucine as an activity unit (U). Mixing the above solutions, digesting in a shaker at 37 deg.C and rotation speed of 180 rpm for 1 hr to convert more than 95% of tissue into cell suspension, filtering the cell suspension to remove impurities, mixing, and counting. Take 1X 106Adding flow antibody anti-mouse F4/80 and CD11b into each cell, incubating for 1 hour in a dark place, and detecting F4/80 in single cell suspension by flow cytometry+CD11b+The cell ratio is the ratio of tumor-associated macrophages. The ratio was multiplied by the total number of tumor cells per mouse to determine the absolute number of macrophages.
2.7 Effect of macrophage targeting immune preparations on the tumor-associated macrophage ability to sphere.
The established tumor-bearing mice are taken from 5 mice per group, sacrificed, the tumor on the right waist and back is stripped, sheared, 2 times of volume of collagenase A (1mg/mL) is added into the sheared tumor tissue of each mouse, the enzyme activity is 0.223U/mg, the enzyme activity is defined as acting on natural collagen at 37 ℃ and ph7.5, and the amount of polypeptide released from the collagen per hour is equivalent to that of ninhydrin developing 1 micromole leucine as an activity unit (U). Mixing the above solutions, standing at 37 deg.C, rotating at the speed ofDigesting for 1 hour in a shaking table at 180 r/min, converting more than 95% of tissues into cell suspension, filtering the cell suspension to remove impurities, mixing uniformly, and counting. Sorting by flow cytometer to obtain F4/80+CD11b+A cell. The macrophage was inoculated into a 24-well plate at 1000/well, 1mL of stem cell medium and 100ng of M-CSF were added per well, cultured at 37 ℃ for 7 days, the number of cell balls with a diameter of 70 μ M or more was measured by an inverted fluorescence microscope, and the cell balling rate was calculated.
2.8 Effect of macrophage targeting immune preparations on the expression of tumor-associated macrophage sternness factor.
Preparation with 2.7 gave F4/80+CD11b+A cell. Take 1X 106And (3) extracting RNA from the cells, and detecting the expression condition of the xerosis related factor by a qPCR method.
3. Results of the experiment
3.1 preparation and characterization of TAM-Bi formulations.
As a result of the examination (FIGS. 1 and 2), it was found that the liposome (TAM-Bi) coated with the Mp and Bi dipeptides had a particle size of 108.30. + -. 3.61nm, PDI of 0.277 and a potential of-14.80. + -. 2.42 mV. The peak area of the liposome equally coated with Mp and Bi before dialysis was 6324276, the solution in the dialysis bag did not increase after dialysis, and the peak area was 5164960 after dialysis. The encapsulation efficiency was found to be 81.67%. Detecting that the pH value is 3.5, and the potential of the blank liposome is-8.76 +/-1.54 mV; the macrophage targeting peptide (Mp) was mixed with the liposome solution to prepare TAM-Con, and the potential was measured to be-5.74. + -. 0.85 mV. From the above results, the surface potential of the liposome was increased by about 3mV, indicating that Mp was supported on the liposome surface. Since tryptophan (W) in Mp has fluorescent properties, iodide ions can quench their fluorescence. And the added iodide ions in the TAM-Bi make the TAM-Bi difficult to pass through the phospholipid bilayer, so that the contact chance of tryptophan embedded in the bilayer and the iodide ions is greatly reduced, and quenching is difficult to occur. From the analysis of the results, the macrophage targeting peptide is successfully embedded into the liposome coated with the Bcl6 inhibitory peptide, namely, the construction of the dual targeting TAM-Bi is successful.
3.2 characterization of entry of microspheroidal particles into macrophages.
Blank liposome (control group) and rhodamine-loaded macrophage targeting immunoregulation preparation (experimental group) are respectively used for acting on the mouse lung cancer cell model. Research shows that (figure 3) the percentage of cells expressing rhodamine in the TAM of the experimental group of mice is as high as 78.3%. The microsphere particle loaded with the target molecule inhibiting peptide can be effectively and specifically taken up by macrophage.
3.3 Bcl6 expression of TAMs and its effect on dryness of TAMs.
Whether the transcription factor Bcl6 is expressed in TAMs of lung cancer mice is detected by adopting western blot, the result shows that (figure 4) the Bcl6 is highly expressed in the TAMs, and the expression of the Bcl6 is further interfered by adopting small molecular RNA of the Bcl6, so that the expression levels of the dryness factor such as klf2, klf4, myc, nanog, oct4, sox2 and the like in an experimental group are obviously lower than those of a control group. Indicating that high expression of Bcl6 in TAMs can promote differentiation of TAMs into a type with stem cell-like properties.
3.4 effects of TAM-Bi on tumors in lung cancer mice.
TAM-Con (control group) and targeted inhibitor liposome TAM-Bi (treatment group) are respectively acted on a mouse lung cancer cell model. The study showed (figure 5) that the control mice generated tumors of greater volume and weight than the treated mice, which was statistically significant. The macrophage targeting immune preparation TAM-Bi can obviously inhibit the generation and development of tumors.
3.5 Effect of TAM-Bi on macrophage number and proportion.
The number of tumor macrophages in the control group and the treated group was measured by flow-based assay, and the results showed (FIG. 6) that the tumor of TAM-Con-treated control mice was CD11b+F4/80+The proportion and the quantity of macrophages are obviously higher than those of the TAM-Bi treatment group, which shows that the TAM-Bi can obviously reduce the quantity of macrophages in tumors and suggests that the macrophage targeting immune preparation can influence the tumorigenesis and development by inhibiting cancer-promoting macrophages.
3.6 effects of TAM-Bi on macrophage self-renewal and sternness transformation.
Macrophages with self-renewal ability (self-renewal) have long retention time in vivo, fast proliferation and obvious cancer promotion effect, and the influence of TAM-Bi on the self-renewal ability of macrophages is detected by a cell balling-up experiment (sphenoid-forming test). The results show (FIG. 7) that tumor macrophages from TAM-Bi-treated mice formed stem cell-like spheroids in much lower numbers and sizes than those of TAM-Con-treated mice. Since the cell self-renewal capacity is an important functional index of dry transformation of cells, the level of the stem cell marker molecules expressed by macrophages is further detected. The results show (FIG. 8) that TAMs in TAM-Bi treated group express drying factors such as klf2, klf4, myc, nanog, oct4, sox2, etc. at levels significantly lower than those in control cells.
In conclusion, the invention provides an immune microsphere preparation based on macrophage and transcription regulatory molecule Bcl6 double targeting, a preparation method and application in antitumor treatment. The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention; the application of the invention is not limited to tumors, and all the components which are not clear in the embodiment can be realized by the prior art and expanded to other abnormal pathologies or disease prevention and treatment.
Sequence listing
<110> Nanjing university of traditional Chinese medicine
<120> immunoregulation microsphere preparation targeting tumor-associated macrophages, and preparation method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> macrophage targeting peptide (Mp)
<400> 1
Tyr Glu Gln Asp Pro Trp Gly Val Lys Trp Trp Tyr
1 5 10
<210> 2
<211> 9
<212> PRT
<213> B cell lymphoma 6 factor (Bcl6)
<400> 2
Gly Glu Gly Ile Glu His Ile Ser Arg
1 5
Claims (4)
1. The application of the immunoregulation microsphere preparation targeting tumor-associated macrophages in preparing anti-tumor products is characterized in that the microsphere preparation is a dual-targeting preparation combining macrophage targeting peptide and B cell lymphoma 6 factor inhibitory peptide; wherein the tumor is lung cancer; wherein the amino acid sequence of the macrophage targeting peptide is shown as YEQDPWGVKWWY in SEQ ID No.1, and the amino acid sequence of the B cell lymphoma 6 factor inhibiting peptide is shown as GEGIEHISR in SEQ ID No. 2;
the preparation method of the immunoregulation microsphere preparation targeting tumor-associated macrophages comprises the following steps:
(i) loading B cell lymphoma 6 factor inhibitory peptide into liposome, namely dissolving the B cell lymphoma 6 factor inhibitory peptide into PBS buffer solution, adding a phospholipid film, and oscillating to obtain B cell lymphoma 6 factor inhibitory peptide-liposome solution;
(ii) combining the B cell lymphoma 6 factor inhibitory peptide-liposome solution with macrophage targeting peptide to obtain the final product;
in step (i), the amount of the phospholipid membrane is controlled so that the molar ratio of the phospholipid to the B-cell lymphoma 6 factor inhibitory peptide is 40:1-8: 5;
in step (ii), said binding the B-cell lymphoma 6 factor inhibitory peptide-liposome solution with the macrophage targeting peptide comprises the steps of:
(1) dissolving macrophage targeting peptide in PBS buffer solution, and adjusting pH to 2-4;
(2) and (2) mixing the solution obtained in the step (1) with a B cell lymphoma 6 factor inhibitory peptide-liposome solution to obtain the compound.
2. The use of claim 1, wherein the ratio of the amount of the B-cell lymphoma factor 6 inhibitory peptide to the amount of the PBS buffer is 1-4 mg/mL.
3. The use of claim 1, wherein in step (1), the amount ratio of the macrophage targeting peptide to the PBS buffer is 1-4 mg/mL.
4. The use according to claim 1, wherein in the step (2), the amounts of the solution obtained in the step (1) and the B-cell lymphoma 6 factor inhibiting peptide-liposome solution are controlled so that the molar ratio of the macrophage targeting peptide to the phospholipid is 1:50-1: 200.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102573500A (en) * | 2009-08-06 | 2012-07-11 | 纽拉尔图斯制药公司 | Treatment of macrophage-related disorders |
| CN103998935A (en) * | 2011-04-28 | 2014-08-20 | 斯隆-凯特林癌症研究所 | Hsp90 combination therapy |
| CN106573070A (en) * | 2014-08-13 | 2017-04-19 | 约翰霍普金斯大学 | Selective dendrimer delivery to brain tumors |
| CN107573418A (en) * | 2017-08-21 | 2018-01-12 | 华中科技大学 | Tumor-associated macrophage double target polypeptide, nano particle, preparation and applications |
| CN108064295A (en) * | 2015-01-14 | 2018-05-22 | 埃克西奎雷股份有限公司 | Nucleic acid nano structure with core motif |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2020511997A (en) * | 2017-03-31 | 2020-04-23 | ザイムワークス，インコーポレイテッド | Tumor antigen presentation inducer construct and use thereof |
-
2020
- 2020-04-21 CN CN202010316985.4A patent/CN111467472B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102573500A (en) * | 2009-08-06 | 2012-07-11 | 纽拉尔图斯制药公司 | Treatment of macrophage-related disorders |
| CN103998935A (en) * | 2011-04-28 | 2014-08-20 | 斯隆-凯特林癌症研究所 | Hsp90 combination therapy |
| CN106573070A (en) * | 2014-08-13 | 2017-04-19 | 约翰霍普金斯大学 | Selective dendrimer delivery to brain tumors |
| CN108064295A (en) * | 2015-01-14 | 2018-05-22 | 埃克西奎雷股份有限公司 | Nucleic acid nano structure with core motif |
| CN107573418A (en) * | 2017-08-21 | 2018-01-12 | 华中科技大学 | Tumor-associated macrophage double target polypeptide, nano particle, preparation and applications |
Non-Patent Citations (3)
| Title |
|---|
| Inhibitory Effect of Purpurogallin on Osteoclast Differentiation In Vitro through the Downregulation of c-Fos and NFATc1;Kiryeong Kim等;《International journal of molecular sciences》;20180217;第19卷(第2期);601 * |
| Novel therapeutic interventions in cancer treatment using protein and peptide-based targeted smart systems;SobiyaZafar等;《Seminars in Cancer Biology》;20190820;第12页右栏第4段，第14页右栏第3段 * |
| 氯氰菊酯对巨噬细胞的毒性作用和极化的影响及其相关机制;黄芳;《中国博士学位论文全文数据库医药卫生科技辑》;20170615(第06期);E055-2 * |
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Application publication date: 20200731 Assignee: Zhengzhou Bain Biotechnology Co.,Ltd. Assignor: Zhengzhou Yuanchuang Gene Technology Co.,Ltd.|Henan Yuanchuang life stem cell bank technology Co.,Ltd. Contract record no.: X2023980034401 Denomination of invention: An immunomodulatory microsphere formulation targeting tumor associated macrophages and its preparation method and application Granted publication date: 20201225 License type: Common License Record date: 20230403 |